Development And Application Of SSR Primers Based On RAD-Seq Data Of Panax Bipinnatifidus Complex

The unique advantages of molecular markers are irreplaceable in species identification and biodiversity research.Especially,the simple sequence repeat(SSR)markers are mendelian inheritance and co-dominantssr,the results are remarkable stability and polymorphism,and not strict in quality and quantity requirements for DNA,so that they have been widely developed and used in various organism.In this study,we focus on the development and application of Panax bipinnatifidus complex.The Panax bipinnatifidus complex comprises many species,belonging to the genus Panax L.(family Araliaceae),is famous herbal medicine in Asia.However,because there are many transitional characters in root,leaf,flower and fruit of this complex group,secondly,have experienced short-term adaptive radiation,the taxonomic category and status of this complex group in ginseng genus have been long-term controversial.We used the Mi dd RAD-seq(a modified double digest restriction-site associated DNA sequencing technology)data from 29 individuals of Panax bipinnatifidus complex with samples of tetraploid specie P.zingiberensis excluded,to develop SSR primers.Then the 11 primers successfully developed were used to verify the polymorphism in 66 individuals of Panax bipinnatifidus complex collected from 22 locations,and test the universality in other three species(P.notoginseng,P.japonicus,P.stipuleanatus)from panax.The SSR primers developed in this study will help us to carry out further studies on the taxonomic and genetic diversity of Panax bipinnatifidus complex.The main results are as follows:(1)We had scavenged SSR markers from MI dd RAD-seq data of 29 individuals of Panax bipinnatifidus complex by Stacks and QDD software package.Distribution and composition characteristics of SSR site in Panax bipinnatifidus complex were statistically analyzed by Excel.We obtained 7458 sequences containing pure SSRs sites in this study,in which repetition unit type,repetition sequence length and repetition number were negatively corelated with the number of SSRs sites.There are more dinucleotide repetition SSRs sites than trinucleotide SSRs repetition sites,among which the dominant repetition motifs are AG/CT and AGC/CTG,The length of SSR sequence is mainly 10-25 bp,in which the largest number of SSRs is 12 bp(24.48%)in length,followed by 18 bp(21.31%).(2)SSRs primers were designed according to the flanking sequences.Totally 114 primers were selected in 7458 sequences containing pure SSRs sites by establishing 9primer screening criteria.And 11 pairs of SSR primers were screened and remained because of high successful PCR amplified rate(97.52%)and polymorphism(6.91 alleles per pair)analyzed by agarose gel electrophoresis and capillary electrophoresis After that,we detected the primer universality of these 11 SSRs primers in 13 individuals from outgroups P.notoginseng,P.stipuleanatus,and P.quinquefolius.The results showed that 10 pairs of primers were successfully amplified and polymorphic in all three species with the rest one primer pair successfully amplified only in P.quinquefolius.Primer P5 and P9 had ideal taxonomic resolution at the species level in the outgroups.There was good polymorphism of primer P7 between individuals in the outgroups.(3)The third work was using the 11 pairs of primers to study and discuss the taxonomic status of samples from Hunan(HN)and Hubei(HB)province.The results indicated that they are probably tetraploidy and could be treated as a genetic unit according to the results of clustering analysis.However,the samples from Hunan(HN)and Hubei(HB)are different taxon from the tetraploidy species P.japanicus distributed in Japan,which used to be treated as one member of Panax bipinnatifidus complex.Futhermore,the samples from Hunan(HN)and Hubei(HB)belong to the Panax bipinnatifidus complex revealed by the clustering analysis,but they do not belong to P.zingiberensis or P.vietnamensis.(4)With the amplification data of 11 SSRs primer pairs of 66 individuals from Panax bipinnatifidus complex,we carried out UPGMA tree and STRUCTURE clustrring analysis.The results indicated that P.zingiberensis and P.vietnamensis were supported to be monophyletic,respectively.In other words,the application of 11 SSRs primers could identify P.zingiberensis and P.vietnamensis.The samples of P.wangianus and P.bipinnatifidus did not form monophyletic clades and but nested into each other.On balance,we mining information from 29 individuals RAD-seq for P.bipinnatifidus complex to calculate the distribution characteristics of SSR site.Then,we develop 11 paris of SSR primers frome this SSR sites.P.zingiberensis and P.vietnamensis could be distinguished for Panax bipinnatifidus complexthe by 11 paris of SSR primers,moreover show a better polymorphism.Secondly,this 11 paris of SSR primers exhibit satisfactory cross-species amplification capacity,they have successfully amplified in the other three species(P.notoginseng,P.stipuleanatus,and P.quinquefolius)of Panax.During the screening of primers,a total of 28 pairs of primers have signals in the f Panax bipinnatifidus complexthe,which can enrich the SSR primer library of Panax.